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Diphtheria Toxin (diphtheria + toxin)
Selected AbstractsSelective Inhibition of Hepatoma Cells Using Diphtheria Toxin A under the Control of the Promoter/Enhancer Region of the Human ,-Fetoprotein GeneCANCER SCIENCE, Issue 3 2000Michito Kunitomi We constructed a plasmid containing human ,-fetoprotein (AFP) promoter/enhancer to direct the cell type-specific expression of diphtheria toxin fragment A (DTA), designated as pAF-DTA, to AFP-producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE-C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF-DTA was transfected, the growth of AFP-positive HuH-7 cells was inhibited, whereas growth inhibition was not observed in AFP-negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF-DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF-DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH-7 and splenic stromal cells were co-cultured revealed the growth inhibition by pAF-DTA to be selective in HuH-7 cells. Finally, the growth of HuH-7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF-DTA/liposome complex into a tumor mass. These results suggest that use of pAF-DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells. [source] Evaluation of combined gene regulatory elements for transcriptional targeting of suicide gene expression to malignant melanomaEXPERIMENTAL DERMATOLOGY, Issue 6 2003Heike Rothfels Abstract:, Selective killing of tumors can be achieved by targeting the transcription of suicide genes via specific DNA control elements to malignant cells. Three different enhancer-promoter systems were constructed and evaluated for their capability to direct gene expression to melanoma. Two tissue-specific (tyrosinase and MIA) promoters and one weak viral promoter were fused to multiple tandem copies of a melanocyte-specific enhancer element. Reporter gene assays revealed a maximum increase in transcription by combining each promoter with 3,4 copies of the enhancer and demonstrated that all enhancer-promoter combinations exhibited tissue-specific activity. Though this activity was still significantly less than that of the strong but unspecific cytomegalo virus (CMV) promoter. In contrast, when these combinations were employed to drive the expression of two suicide genes, encoding the diphtheria toxin A chain (DT-A) and the prodrug-activating herpes simplex virus thymidine kinase (TK), respectively, only those constructs in which transcription was under the control of tissue-specific promoter elements mediated selective killing of melanoma cells. This killing was in the range of cell death induced by CMV promoter activity. Our data indicate that the enhancer/tyrosinase and enhancer/MIA promoter constructs but not the viral promoter constructs can provide a valuable tool for selective suicide gene expression in melanoma. [source] Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugateFEBS JOURNAL, Issue 18 2007Sandra E. Geden Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments. [source] Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentumJOURNAL OF NEUROCHEMISTRY, Issue 1 2007S. D. Clark Abstract Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 , 30 nmol/L; calcium mobility assay) and to be 10 000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 , 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures. [source] Enhancement of Diphtheria Toxin Potency by Replacement of the Receptor Binding Domain with Tetanus Toxin C-FragmentJOURNAL OF NEUROCHEMISTRY, Issue 6 2000A Potential Vector for Delivering Heterologous Proteins to Neurons Abstract: This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB389TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB389) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB389TTC was , 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB389MSH. The cytotoxic effect of DAB389TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB389TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB389 catalytic fragment. These results suggest that a catalytically inactive form of DAB389TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons. [source] Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccinesPEDIATRIC ALLERGY AND IMMUNOLOGY, Issue 1 2008Christina E. West Microbial exposure is necessary for the development of normal immune function, which has driven the idea of using probiotics for treatment and prevention of immune-mediated diseases in infancy and childhood. Mounting evidence indicates that probiotics have immunomodulatory effects. However, the mechanisms are still poorly understood. Specific antibody response is a valuable proxy for immune system maturation status in infancy. We aimed at determining the impact of Lactobacillus F19 (LF19) during weaning on infections and IgG antibody responses to routine vaccines. In a double-blind, placebo-controlled randomized intervention trial, infants were fed cereals with (n = 89) or without LF19 (n = 90) from 4 to 13 months of age. Infants were immunized with DTaP (diphtheria and tetanus toxoid and acellular pertussis), polio and Hib-conjugate vaccines at (3), 5 and 12 months of age. We assessed the number of days with infections, antibiotic prescriptions and antibody concentrations to Hib capsular polysaccharide (HibPS), diphtheria toxin (D) and tetanus toxoid (T) before and after the second and third doses. Days with infectious symptoms did not differ between the groups. Days with antibiotic prescriptions were fewer in the LF19 group (p = 0.044). LF19 enhanced anti-D concentrations when adjusting for breastfeeding duration and colonization with LF19 (p = 0.024). There was an interaction of the intervention and colonization with LF19 on anti-T concentrations during the course of vaccination (p = 0.035). The anti-HibPS concentrations were higher after the first and second dose of Hib vaccine in infants breastfed <6 months compared with those breastfed ,6 months (p < 0.05), with no effect by LF19. In conclusion, feeding LF19 did not prevent infections, but increased the capacity to raise immune responses to protein antigens, with more pronounced effects in infants breastfed <6 months. [source] Regulatory sequences of H19 and IGF2 genes in DNA-based therapy of colorectal rat liver metastasesTHE JOURNAL OF GENE MEDICINE, Issue 3 2005Patricia Ohana Abstract Background Malignant tumors of the liver are among the most common causes of cancer-related death throughout the world. Current therapeutic approaches fail to control the disease in most cases. This study seeks to explore the potential utility of transcriptional regulatory sequences of the H19 and insulin growth factor 2 (IGF2) genes for directing tumor-selective expression of a toxin gene (A fragment of diphtheria toxin), delivered by non-viral vectors. Methods The therapeutic potential of the toxin vectors driven by the H19 and the IGF2-P3 regulatory sequences was tested in a metastatic model of rat CC531 colon carcinoma in liver. Results Intratumoral injection of these vectors into colon tumors implanted in the liver of rats induced an 88% and a 50% decrease respectively in the median tumor volume as compared with the control groups. This therapeutic action was accompanied by increased necrosis of the tumor. Importantly, no signs of toxicity were detected in healthy animals after their treatment by the toxin expression vectors. Conclusions DT-A was preferentially expressed in liver metastases after being transfected with H19 or IGF2-P3 promoter-driven DT-A expression plasmids, causing a very significant inhibition of tumor growth as a result of its cytotoxic effect. Our findings strongly support the feasibility of our proposed therapeutic strategy, which may contribute to open new gene therapeutic options for human liver metastases. Copyright © 2004 John Wiley & Sons, Ltd. [source] Muscle resident macrophages control the immune cell reaction in a mouse model of notexin-induced myoinjuryARTHRITIS & RHEUMATISM, Issue 1 2010Madly Brigitte Objective Skeletal muscle may be the site of a variety of poorly understood immune reactions, particularly after myofiber injury, which is typically observed in inflammatory myopathies. This study was undertaken to explore both the cell dynamics and functions of resident macrophages and dendritic cells (DCs) in damaged muscle, using a mouse model of notexin-induced myoinjury to study innate immune cell reactions. Methods The myeloid cell reaction to notexin-induced myoinjury was analyzed by microscopy and flow cytometry. Bone marrow (BM) transplantation studies were used to discriminate resident from exudate monocyte/macrophages. Functional tests included cytokine screening and an alloantigenic mixed leukocyte reaction to assess the antigen-presenting cell (APC) function. Selective resident macrophage depletion was obtained by injection of diphtheria toxin (DT) into CD11b,DT receptor,transgenic mice transplanted with DT-insensitive BM. Results The connective tissue surrounding mouse muscle/fascicle tissue (the epimysium/perimysium) after deep muscle injury displayed a resident macrophage population of CD11b+F4/80+CD11c,Ly-6C,CX3CR1, cells, which concentrated first in the epimysium. These resident macrophages were being used by leukocytes as a centripetal migration pathway, and were found to selectively release 2 chemokines, cytokine-induced neutrophil chemoattractant and monocyte chemoattractant protein 1, and to crucially contribute to massive recruitment of neutrophils and monocytes from the blood. Early epimysial inflammation consisted of a predominance of Ly-6ChighCX3CR1lowCD11c, cells that were progressively substituted by Ly-6ClowCX3CR1high cells displaying an intermediate, rather than high, level of CD11c expression. These CD11cintermediate cells were derived from circulating CCR2+ monocytes, functionally behaved as immature APCs in the absence of alloantigenic challenge, and migrated to draining lymph nodes while acquiring the phenotype of mature DCs (CD11c+Ia+CD80+ cells, corresponding to an inflammatory DC phenotype). Conclusion The results in this mouse model show that resident macrophages in the muscle epimysium/perimysium orchestrate the innate immune response to myoinjury, which is linked to adaptive immunity through the formation of inflammatory DCs. [source] Structures of three diphtheria toxin repressor (DtxR) variants with decreased repressor activityACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2001Ehmke Pohl The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae regulates the expression of the gene on corynebacteriophages that encodes diphtheria toxin (DT). Other genes regulated by DtxR include those that encode proteins involved in siderophore-mediated iron uptake. DtxR requires activation by divalent metals and holo-DtxR is a dimeric regulator with two distinct metal-binding sites per three-domain monomer. At site 1, three side chains and a sulfate or phosphate anion are involved in metal coordination. In the DtxR,DNA complex this anion is replaced by the side chain of Glu170 provided by the third domain of the repressor. At site 2 the metal ion is coordinated exclusively by constituents of the polypeptide chain. In this paper, five crystal structures of three DtxR variants focusing on residues Glu20, Arg80 and Cys102 are reported. The resolution of these structures ranges from 2.3 to 2.8,Å. The side chain of Glu20 provided by the DNA-binding domain forms a salt bridge to Arg80, which in turn interacts with the anion. Replacing either of the salt-bridge partners with an alanine reduces repressor activity substantially and it has been inferred that the salt bridge could possibly control the wedge angle between the DNA-binding domain and the dimerization domain, thereby modulating repressor activity. Cys102 is a key residue of metal site 2 and its substitution into a serine abolishes repressor activity. The crystal structures of Zn-Glu20Ala-DtxR, Zn-Arg80Ala-DtxR, Cd-Cys102Ser-DtxR and apo-Cys102Ser-DtxR in two related space groups reveal that none of these substitutions leads to dramatic rearrangements of the DtxR fold. However, the five crystal structures presented here show significant local changes and a considerable degree of flexibility of the DNA-binding domain with respect to the dimerization domain. Furthermore, all five structures deviate significantly from the structure in the DtxR,DNA complex with respect to overall domain orientation. These results confirm the importance of the hinge motion for repressor activity. Since the third domain has often been invisible in previous crystal structures of DtxR, it is also noteworthy that the SH3-like domain could be traced in four of the five crystal structures. [source] Humanized immunotoxins: A new generation of immunotoxins for targeted cancer therapyCANCER SCIENCE, Issue 8 2009Mrudula Mathew Chemotherapy, radiation, and surgery are the conventional treatment modalities for cancer. The success achieved with these approaches has been limited due to several factors like chemoresistance to drugs, non-specificity leading to peripheral toxicity, and non-resectable tumors. To combat these problems, the concept of targeted therapy using immunotoxins was developed. Immunotoxins are chimeric proteins with a cell-selective ligand chemically linked or genetically fused to a toxin moiety and can target cancer cells overexpressing tumor-associated antigens, membrane receptors, or carbohydrate antigens. Ligands for these receptors or monoclonal antibodies or single chain variable fragments directed against these antigens are fused with bacterial or plant toxins and are made use of as immunotoxins. Pseudomonas exotoxin, anthrax toxin, and diphtheria toxin are the commonly used bacterial toxins. Ricin, saporin, gelonin, and poke weed antiviral protein are the plant toxins utilized in immunotoxin constructs. Several such fusion proteins are in clinical trials, and denileukin difitox is a FDA-approved fusion protein. In spite of the promise shown by bacterial- and plant toxin-based chimeric proteins, their clinical application is hampered by several factors like immunogenicity of the toxin moiety and non-specific toxicity leading to vascular leak syndrome. In order to overcome these problems, a novel generation of immunotoxins in which the cytotoxic moiety is an endogenous protein of human origin like proapoptotic protein or RNase has been developed. This review summarizes the advances in this new class of fusion protein and the future directions to be explored. (Cancer Sci 2009) [source] Selective Inhibition of Hepatoma Cells Using Diphtheria Toxin A under the Control of the Promoter/Enhancer Region of the Human ,-Fetoprotein GeneCANCER SCIENCE, Issue 3 2000Michito Kunitomi We constructed a plasmid containing human ,-fetoprotein (AFP) promoter/enhancer to direct the cell type-specific expression of diphtheria toxin fragment A (DTA), designated as pAF-DTA, to AFP-producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE-C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF-DTA was transfected, the growth of AFP-positive HuH-7 cells was inhibited, whereas growth inhibition was not observed in AFP-negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF-DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF-DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH-7 and splenic stromal cells were co-cultured revealed the growth inhibition by pAF-DTA to be selective in HuH-7 cells. Finally, the growth of HuH-7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF-DTA/liposome complex into a tumor mass. These results suggest that use of pAF-DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells. [source] The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricinCELLULAR MICROBIOLOGY, Issue 8 2008Pradeep K. Gupta Summary Diphthamide is a post-translational derivative of histidine in protein synthesis elongation factor-2 (eEF-2) that is present in all eukaryotes with no known normal physiological role. Five proteins Dph1,Dph5 are required for the biosynthesis of diphthamide. Chinese hamster ovary (CHO) cells mutated in the biosynthetic genes lack diphthamide and are resistant to bacterial toxins such as diphtheria toxin. We found that diphthamide-deficient cultured cells were threefold more sensitive than their parental cells towards ricin, a ribosome- inactivating protein (RIP). RIPs bind to ribosomes at the same site as eEF-2 and cleave the large ribosomal RNA, inhibiting translation and causing cell death. We hypothesized that one role of diphthamide may be to protect ribosomes, and therefore all eukaryotic life forms, from RIPs, which are widely distributed in nature. A protective role of diphthamide against ricin was further demonstrated by complementation where dph mutant CHO cells transfected with the corresponding DPH gene acquired increased resistance to ricin in comparison with the control-transfected cells, and resembled the parental CHO cells in their response to the toxin. These data show that the presence of diphthamide in eEF-2 provides protection against ricin and suggest the hypothesis that diphthamide may have evolved to provide protection against RIPs. [source] | ||||||||||||||||